Bielecki J
Institute of Microbiology, Warsaw University, Poland.
Acta Microbiol Pol. 1994;43(2):133-43.
To carry out efficient insertional mutagenesis in Listeria monocytogenes 1040S and to facilitate the characterisation of disrupted genes, three novel derivatives of plasmid pACYC 184 were constructed, p-LIV 1, pLIV-2 and pLIV-3. The technique is simple and rapid and can be applied to most genes, even those that are essential. The method is unique and particularly effective by the use of a temperature-sensitive pE 194 replicon to facilitate the insertion of the gene. After transformation of the plasmid into L. monocytogenes it is possible to select for integration of the plasmid into the chromosome at 42 degrees C. High insertion frequency and convenience for looking for specific mutations with known sites of insertion make them the plasmid derivatives of choice for insertional mutagenesis in any bacteria that support replication of pE 194 TS. Three insertional mutants of L. monocytogenes are described. Two insertions were shown to be within iap gene, one in hly gene. The supernatants and the pellets from the iap mutants had no detectable haemolytic titer when assayed without the reducing agent.
为了在单核细胞增生李斯特菌1040S中进行高效的插入诱变，并便于对破坏的基因进行表征，构建了质粒pACYC 184的三种新型衍生物，即p-LIV 1、pLIV-2和pLIV-3。该技术简单快速，可应用于大多数基因，甚至是必需基因。该方法独特，通过使用温度敏感的pE 194复制子促进基因插入，效果尤为显著。将质粒转化到单核细胞增生李斯特菌后，可在42℃下选择质粒整合到染色体中。高插入频率以及便于寻找具有已知插入位点的特定突变，使其成为在任何支持pE 194 TS复制的细菌中进行插入诱变的首选质粒衍生物。描述了单核细胞增生李斯特菌的三个插入突变体。两个插入显示在iap基因内，一个在hly基因内。在没有还原剂的情况下进行检测时，iap突变体的上清液和沉淀没有可检测到的溶血效价。
